Formation of metaphase chromosome is essential for higher eukaryotes to ensure proper and accurate segregation of genomic information into two daughter cells. In order to clarify the structural basis of chromosome formation, we have carried out proteome analyses of human metaphase chromosomes and identified more than two hundreds of chromosome proteins. Comparative proteome analyses using the chromosomes from different preparation methods as well as from different cell lines eliminated proteins that are not essential for the higher order structure of chromosomes. Different types of electrophoresis were employed for the efficient and high-resolution separation of constituent proteins. Furthermore, absence of chromosome titin, which is a giant protein with the molecular mass of 3MDa and had been reported as a protein contributing to chromosome higher-order structure, was confirmed by proteomic analysis in addition to transcript analysis. Finally we established a chromosome four layer model where all chromosome proteins are classified into one of four different groups. Our recent functional analyses on chromosome proteins in vivo suggest the proteins in the different groups have different roles.
